The invention discloses a test strip for quantitatively detecting contents of a total prostate specific antigen and a free prostate specific antigen simultaneously through adopting a time-resolved fluorescent immunochromatographic method as well as a preparation method and a test paper card. The test strip comprises a bottom board, absorbent paper, a nitrocellulose membrane, a combining pad and a sample pad, wherein the absorbent paper, the nitrocellulose membrane, the combining pad and the sample pad are sequentially adhered to the bottom board; a first inspection band of an fPSA membrane scratching antibody, a second inspection band of a complex PSA membrane scratching antibody and a quality control band of a rabbit antimouse polyclonal antibody are sequentially arranged on the nitrocellulose membrane at intervals in parallel, the surface of the combining pad is coated with a composite prostate-specific antigen antibody and a free prostate specific antigen antibody, and both the composite prostate-specific antigen antibody and the free prostate specific antigen antibody are coupled with nano microspheres with lanthanum rare earth ions, so that the high detection result accuracy and small detection result errors of the tPSA and the fPSA in same sample liquid are guaranteed, and great convenience is provided for clinical application.